三角褐指藻岩藻黄素合成途径及其关键基因对高光照的响应

以三角褐指藻为材料,以岩藻黄素的合成途径中主要的六个酶:八氢番茄红素合成酶、八氢番茄红素脱氢酶、ζ-胡萝卜素脱氢酶、胡萝卜素异构酶、番茄红素β-环化酶和玉米黄素环氧酶为研究对象,研究高光照胁迫对三角褐指藻岩藻黄素合成代谢途径中关键基因表达量以及岩藻黄素含量的变化规律。以光照强度为500μE/(m2·s),分别照射处理三角褐指藻3h、6h、12h,采用HPLC对岩藻黄素含量进行定量分析。随着光照时间的延长,岩藻黄素含量呈现先上升后下降的趋势;高光照6h样品中岩藻黄素含量为1.85mg·g-1DW(dry cell weight),比高光照0h对照组中岩藻黄素含量增加了2.16倍。qRT-PCR分析结果表明玉米黄素环氧酶基因zep1和八氢番茄红素合成酶基因pys转录本在高光照胁迫6h时丰度最高,与对照组相比提高了近2倍,基因表达量的变化表现出与岩藻黄素含量变化相一致的趋势。高光照处理3h后,胡萝卜素异构酶基因crtiso3、八氢番茄红素脱氢酶基因pds、ζ-胡萝卜素脱氢酶基因zds的表达水平相比于对照组明显降低,表现出应对高光照胁迫的快速响应;番茄红素β-环化酶基因lcyb、胡萝卜素异构酶基因crtiso1的表达水平则在高光照处理12h后明显提高;与zep1和pys相比,lcyb和crtiso1基因的表达对高光照胁迫处理的响应时间要滞后至少6h。本研究揭示了高光照胁迫处理下三角褐指藻中岩藻黄素含量及其合成途径中关键基因的表达模式,表明zep1和pys基因表达水平与岩藻黄素合成量之间存在明显的线性关系。     

盐度对三角褐指藻生长及有机质积累的影响

以三角褐指藻为材料,探究盐度对该藻生长、总脂等有机质含量的影响,探讨有利于油脂生产的盐度条件。结果显示,盐度变化对三角褐指藻生物量和有机质积累均有显著影响。在盐度70‰的海水中培养的细胞,其比生长速率比正常海水盐度（35‰）培养组降低了22.6%。正常海水盐度下,生物量及总脂含量最高,分别达1.92g/L和29.98%,且总脂单位体积产量极显著（p＜0.01）地高于其他各组,藻油产量高达47.67mg•L-1•d-1。在盐度17‰时,多糖含量最高,达5.5%。在盐度70‰时,蛋白质含量最高,达17.82%。       

花生△12脂肪酸脱氢酶基因AhFAD2B在酿酒酵母中的表达及功能分析

花生油中亚油酸(LA,C18:2Δ9,12)含量很高,为了研究花生Δ12脂肪酸脱氢酶(Δ12 FAD)的功能,用RT-PCR方法从花生未成熟种子中扩增出AhFAD2B的cDNA,连接到酵母表达载体p416中,并转化酿酒酵母K601,得到工程菌Kp4AFAD2B。气相色谱分析脂肪酸成分,结果表明该基因能在酵母K601中表达,并使菌株产生了两种新的脂肪酸即棕榈二烯酸(C16∶2Δ9,12)和亚油酸(C18∶2Δ9,12),含量分别占总脂肪酸的2.1%和9.2%,棕榈油酸(C16∶1Δ9)和油酸(C18∶1Δ9)含量与对照相比相应地下降,证明该基因编码的Δ12脂肪酸脱氢酶除能作用于C18∶1Δ9,还具有催化16碳的C16∶1Δ9底物在Δ12位脱氢生成C16∶2Δ9,12的功能,但在两种底物之间可能更偏爱C18∶1Δ9。     

茶树PPO融合蛋白真核表达载体的构建与表达

以迎霜品种茶树基因组为模板,利用高保真聚合酶pfu,通过PCR方法扩增得到茶树PPO基因的编码区序列。通过设计酶切引物将其成功融合至真核表达载体pPICZa中,构建出茶多酚氧化酶的融合蛋白真核表达载体pPICZa-PPO,并将其转化进入巴斯德毕赤酵母（Pichia pastoris）GS115中,成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导,采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性,能够正确发挥酶蛋白的生物功能。     

橡胶树2个胶乳转化酶基因的原核表达

转化酶是控制橡胶树胶乳蔗糖代谢和影响胶乳(橡胶)产量的关键酶。将已克隆的2个橡胶树胶乳转化酶基因(HbNIN1、HbNIN2)的编码区插入到原核表达载体pET28a上,经酶切和测序鉴定,成功获得了读框准确的转化酶表达载体(pET-HbNIN1和pET-HbNIN2);再将表达载体转化大肠杆菌表达菌株BL21(DE3),对其培养温度、IPTG诱导浓度和培养时间进行优化表达。结果表明,携带2个转化酶基因表达载体(pET-HbNIN1和pET-HbNIN2)的大肠杆菌转化子分别在28℃和37℃条件下,经浓度1.0 mmol/L IPTG诱导6 h实现了目标蛋白(HbNIN1和HbNIN2)的原核高效表达,表达量大于菌体总蛋白的20%,目标蛋白主要以包涵体形式存在。该结果为进一步研究这2种转化酶蛋白的分子特性和抗体制备奠定基础。     

铁皮石斛HDR基因克隆及真菌诱导子对其表达和生物碱含量的影响

以铁皮石斛原球茎为材料,采用同源克隆的方法,成功得到了铁皮石斛Do-HDR基因的全长,并通过q PCR对其相对表达量进行分析。结果表明:Do-HDR基因全长1 784 bp(Gen Bank登录号KJ946381),5′UTR为40 bp,3′UTR为361 bp,开放阅读框为1 382 bp,编码460个氨基酸。生物信息学分析结果表明,该基因编码的蛋白为稳定的亲水蛋白,不具有跨膜结构,不含信号肽,定位于内质网。采用灭活的尖孢镰刀菌菌液作为诱导子,分别以0、100、200、500、1 000 mg/L的浓度培养3 d后测定总生物碱含量,结果表明,总生物碱含量呈现先上升后下降的趋势;当诱导子浓度为200 mg/L时,总生物碱含量达到最大值。在此基础上通过q PCR分析Do-HDR基因的相对表达量,结果发现Do-HDR基因相对表达量在诱导子浓度为200 mg/L时也到达最大值。因此,推测Do-HDR基因与生物碱的合成与积累有关。     

甘蓝型油菜中5个脂酰-ACP硫酯酶基因的克隆与功能初步分析

为了解影响油菜种子含油量的关键基因，从高含油量（HO）和低含油量（LO）甘蓝型油菜中克隆了5个脂 酰-ACP硫酯酶（fatty acyl-ACP thioesterase, FAT）的基因。序列分析表明这5个FAT 基因在HO和LO油菜基因组之 间没有序列差异。5个FAT 基因分别命名为：BnaA.FATA.a、BnaC.FATA.b、BnaA.FATA.c、BnaC.FATB.a 和BnaA.FATB. b。分析表明，HO种子中5个FAT 基因的表达水平显著高于LO种子。异源表达结果显示，5个FAT 基因的所有酵母 转化子的含油量均显著增加，并且它们的脂肪酸组成均发生了变化。将这5个FAT 基因转化到拟南芥中进行进一 步的功能分析。5个转基因材料T1种子含油量均显著增加；BnaA.FATA.a、BnaC.FATA.b、BnaA.FATA.c 转基因株系中 的饱和脂肪酸含量显著降低，而不饱和脂肪酸含量均显著增加；相反，在BnaC.FATB.a 和BnaA.FATB.b 转基因材料 中，饱和脂肪酸含量显著增加，而不饱和脂肪酸含量显著降低。结果表明，这5个FAT 基因不仅可以增加拟南芥含 油量，而且可以改变脂肪酸组成比例。     

转移巴西橡胶树HbCBF1基因提高烟草抗寒能力的研究

通过农杆菌介导的叶盘转化法将巴西橡胶树HbCBF1基因导入到烟草中增强表达。GUS染色和PCR鉴定结果表明,外源DNA片段已成功导入并整合到烟草基因组中。选取3个烟草转基因株系用于进一步鉴定分析,分析表明,转化植株游离脯氨酸含量与对照相比,分别上升了4.2倍、3.1倍和2.5倍,叶绿素A和叶绿素B含量也明显提高。电导率法测定转化烟草的相对存活率分别为19.8%、17.5%、16.3%,高于对照植株的7.7%。以上结果表明,橡胶树HbCBF1基因能够增强非低温驯化植物烟草的抗寒能力,其机理可能是通过促进细胞内容物的积累从而提高细胞的低温防御能力。     

红麻酮脂酰辅酶A合成酶(KCS)酵母双杂交诱饵载体构建及自激活检测

酮脂酰辅酶A合成酶(KCS)是超长链脂肪酸合成过程中的限速酶,参与超长链脂肪酸和蜡质的合成,在植物抗旱和应对生物胁迫反应中发挥重要调控作用。研究采用PCR方法从红麻中(茎皮)分离KCS基因片段,将该基因按正确方式插入酵母双杂交表达质粒pGBKT7中,酶切鉴定正确后,用PEG/Li Ac方法转化酵母MaV203,经抗性筛选后,用X-Gal对转化酵母进行自激活检测。结果表明,KCS基因的DNA结合结构域与酵母GAL4转录因子的上游激活位点(UAS)结合,可激活报告基因半乳糖苷酶的表达。经删除KCS基因DNA binding区域后重新转化酵母,结果表明,不携带KCS基因DNA binding区域的截短蛋白可以作为诱饵蛋白来研究与之相互作用的蛋白。     

制霉菌素对褐飞虱若虫解毒酶、尿酸酶含量的影响

【目的】为明确取食制霉菌素处理的水稻对褐飞虱[Nilaparvata lugens (Stål)]相关生理指标的影响,【方法】以不同浓度制霉菌素处理的水稻饲养褐飞虱,检测虫体内相关解毒酶含量与活性以及尿酸酶基因相对含量的变化。【结果】结果显示,取食制霉菌素处理的水稻,褐飞虱体内芳香基酰胺酶(aromatic amidase,AAD)含量前期下降,后期抑制作用逐渐消失,酶含量回升;虫体内O-脱乙基酶(O-deethyl enzyme,ODEE)含量在72 h的处理时长内显著低于对照;羧酸酯酶(carboxylesterase,CarE)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)的活性在不同浓度制霉菌素的处理下呈现不同的动态变化,高浓度制霉菌素(600 U/mL)处理的褐飞虱种群体内的CarE和GST的活性在处理时间段内均呈现诱导-抑制-诱导的现象,而中(300 U/mL)、低浓度(150 U/mL)处理种群中两种酶则随着处理时间的延长呈上升趋势。取食不同浓度制霉菌素处理水稻的褐飞虱种群体内类酵母共生菌尿酸酶基因在24 h、48 h之后与对照相比均无显著差异。取食48 h后,包括对照在内的4个处理尿酸酶基因相对转录水平均有下调趋势,当处理时长达72 h时,尿酸酶基因相对转录水平呈现上调趋势,但取食不同浓度制霉菌素处理水稻的褐飞虱种群体内含量显著低于对照。【结论】取食不同浓度制霉菌素处理的水稻对褐飞虱3龄若虫的4种主要的解毒酶具有不同的影响;制霉菌素处理可通过减少类酵母共生菌数量从而降低尿酸酶的含量。     

Characteristics, composition and thermal stability of Acacia senegal (L.) Willd. seed oil

The chemical composition, main physicochemical properties and thermal stability of oil extracted from Acacia senegal seeds were evaluated. The oil, moisture and the ash contents of the seeds were 9.80%, 6.92% and 3.82%, respectively. Physicochemical properties of the oil were iodine value, 106.56 g/100 g of oil; saponification value, 190.23 mg KOH/g of oil; refractive index (25 °C), 1.471; unsaponifiable matter, 0.93%; acidity, 6.41% and peroxide value, 5.43 meq. O₂/kg of oil. The main fatty acids in the oil were oleic acid (43.62%) followed by linoleic acid (30.66%) and palmitic acid (11.04%). The triacylglycerols (TAGs) with equivalent carbon number ECN 44 (34.90%) were dominant, followed by TAGs ECN 46 (28.19%), TAGs ECN 42 (16.48%) and TAGs ECN 48 (11.23%). The thermal stability analysed in a normal oxidizing atmosphere showed that the oil decomposition began at 268.6 °C and ended at 618.5 °C, with two stages of decomposition at 401.5 °C and 576.3 °C. According to these results, A. senegal seed oil has physicochemical properties, fatty acids composition and thermal characteristics that may become interesting for specific applications in several segments of food and non-food industries.     

Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.     

一个除草剂安全剂AD-67诱导的水稻基因片段的克隆和分析

为寻找化学诱导表达基因,利用mRNA差异显示方法,从除草剂安全剂AD 67（苯叉酰胺）诱导处理的水稻品种9311的幼苗中筛选分离得到1个诱导表达增强的cDNA片段。该片段在处理后6 h表达明显增强,直到第5天仍然有表达。从poly gel上回收片段并测序,核酸同源序列分析显示它与水稻多个EST和cDNA序列一致性达到100%,氨基酸序列分析表明它与水稻、四膜虫、隐球酵母等真核生物的Yippee基因相似性达60%~84%。序列经延伸得到831 bp的cDNA,由此设计引物并进行RT PCR分析,结果说明该基因在除草剂安全剂AD 67诱导后表达增强。     

茶树△12-脂肪酸去饱和酶基因FAD2和FAD6的克隆与表达分析

本研究在茶树转录组测序的基础上,以铁观音茶树的芽叶为材料,采用RT-PCR技术,克隆了茶树不饱和脂肪酸合成途径中的关键限速酶—△12-FAD(12-脂肪酸去饱和酶)基因的包含完整ORF的cDNA序列(CsFAD2和CsFAD6)。生物信息学分析结果表明,CsFAD2的全长为1 184 bp,其开放阅读框(ORF)长度1 149 bp,编码382个氨基酸,定位于内质网上,其氨基酸序列与油茶FAD2的同源性最高达97%;CsFAD6的全长为1 425 bp,其ORF长度为1 311 bp,编码436个氨基酸,定位于叶绿体上,其氨基酸序列与葡萄FAD6同源性达81%。荧光定量PCR结果表明,铁观音茶树幼苗在4℃低温胁迫处理72 h过程中,这两个基因的表达均受低温的诱导,其表达量随着处理时间的延长而升高,在处理48 h时,表达量水平最高;在100 g·L-1的PEG胁迫处理12 h过程中,这两个基因的表达均受PEG胁迫处理的诱导;在ABA(100μmol·L-1)胁迫处理72 h过程中,在处理6~24 h期间,CsFAD2的表达量显著升高,而CsFAD6的表达不受ABA处理的影响,CsFAD6的表达量在处理72 h时显著降低;在Na Cl(250 mmol·L-1)胁迫72 h过程中,CsFAD2的表达量全程降低,而CsFAD6在处理24~72 h期间表达量显著升高。     

The Marine-Derived Fungus Clonostachys rosea,Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 μM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 μM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.     

Studies on samh seeds (Mesembryanthemum forsskalei Hochst) growing in Saudi Arabia: 2: Chemical composition and microflora of samh seeds

The chemical composition of samh seed have been investigated. Proximate analysis showed a composition of 22.25% protein, 5.7% moisture, 5.6% fat, 4.0% ash, 9.7% crude fiber, and the remainder being total carbohydrates. Mineral element analysis revealed that potassium, magnesium, sodium and calcium were present as the major elements. Iron, manganese, zinc and copper were found at lower levels. However, lead was not detected in the samh seeds. Gas-liquid chromatographic analysis of the methylester of the fatty acids of the samh seeds oil revealed the presence of fourteen fatty acids. Linoleic and oleic acids were the principle unsaturated fatty acids. While palmitic acid was the main saturated fatty acid. Amino acid analysis of the samh seeds showed the presence of seventeen amino acids including eight essential amino acids. Glutamic acid, arginine, and aspartic acid were the major amino acids. Cystine and proline were present in trace amounts. These results some of which have not been reported elsewhere indicate the high nutritional potential of Saudi samh seeds. The total aerobic bacterial count and total sporeformers of seeds were 19×10⁷ and 5×10⁴ cfu/g respectively, thus the enterobacteriaceae,B cereus and yeast and molds were 5×10², 1×10² and 7×10² respectively. The seeds were Staph. free and the samh extract had no antimicrobial effect.     

RT-PCR克隆人血清白蛋白cDNA及其序列分析

为了构建人血清白蛋白cDNA橡胶树乳管特异性表达载体，提取人工流产胎儿肝脏组织总RNA，采用RT—PCR法得到其改良cDNA，T／A测序后，采用NCBI BLASTn软件分析测得的序列及由此推导的氨基酸序列，与NCBI数据库中公布的HSA cDNA及氨基酸序列比较其同源率。结果表明，两者分别达到99．6％和99．5％。这为后期橡胶树乳管特异表达载体的构建和以转基因橡胶树来表达人血清白蛋白的研究打下了坚实的前期技术工作基础。     

接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库的构建与分析

利用Trizol法提取接种PRsv 4 d的番木瓜种苗总RNA,分离纯化mRNA,反转录合成双链cDNA,双链cDNA末端经Pfu-DNA聚合酶补平,与EcoR I接头连接,XhoI酶切消化产生粘端.用Sepharose CL-2B柱分离纯化去除小分子cDNA片段,再与pMyr酵母表达载体连接,转化受体菌XL10-Gold,构建了接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库.所获得的原始文库的克隆总数为1.8×106cfu,重组率为100%,文库滴度为2.6×109cfu/mL.对随机选取的34个克隆进行PCR鉴定,结果表明插入片段长度均大于O.5 kb且集中在1 kb左右.文库质量鉴定结果表明,该文库具有较好的库容量、较高的重组率以及较大的插入片段.     

Characterization of Shrimp Oil from Pandalus borealis by High Performance Liquid Chromatography and High Resolution Mass Spectrometry

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as ¹³C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.